Multicolored particles

ABSTRACT

The present invention relates to a set of polymer particles stained with at least two fluorescent dyes, wherein at least 16 subsets of particles can be resolved on the basis of variable emission from the at least two fluorescent dyes wherein emission from at least one dye derives from a fluorescent dye covalently attached to the particle surface, and wherein all particles in said set of polymer particles can bind a uniform amount of a capture reagent. The invention also relates to a method for the preparation of said set of polymer particles as well as a kit comprising said set of polymer particles. The invention further relates to methods and uses of said set of polymer particles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/995,418, filed Jul. 22, 2008, which was a 35 U.S.C. §371 nationalphase application of International Application No. PCT/NO2006/000269,filed Jul. 10, 2006 and claims the benefit of NO20053373, filed Jul. 11,2005, all of which are incorporated herein by reference as if fully setforth.

FIELD OF THE INVENTION

The present invention relates to a set of polymer particles stained withat least two fluorescent dyes, wherein at least 16 subsets of particlescan be resolved on the basis of variable emission from the at least twofluorescent dyes wherein emission from at least one dye derives from afluorescent dye covalently attached to the particle surface, and whereinall particles in said set of polymer particles can bind a uniform amountof a capture reagent. The invention also relates to a method for thepreparation of said set of polymer particles as well as a kit comprisingsaid set of polymer particles. The invention further relates to methodsand uses of said set of polymer particles.

BACKGROUND OF THE INVENTION

It is recognized that two or more dyes of varying proportions could beused to increase the permutation number of unique combinations of dyesin a single particle. The unique emission wavelengths and fluorescenceintensities could be useful for multiparameter or multiplex analysis ofa plurality of analytes in the same sample.

Three methods of making colored, fluorescent beads have been disclosed,including: (a) covalent attachment of dyes onto the surface of theparticle (e.g. U.S. Pat. No. 4,774,189 Schwartz; U.S. Pat. No. 5,194,300Cheung), (b) internal incorporation of dyes during particlepolymerization (e.g. U.S. Pat. No. 5,073,498 Schwartz; U.S. Pat. No.4,717,655 Fulwyler), and (c) dyeing after the particle has been alreadypolymerized (e.g. L. B. Bangs, Uniform Latex J Particles; SeragenDiagnostics Inc. 1984).

U.S. Pat. No. 5,194,300 Cheung and U.S. Pat. No. 4,774,189 Schwartzdisclose fluorescent microspheres that are coated by covalentlyattaching either one or a plurality of fluorescent dyes to theirsurface. However, the features of the particles do not meet thespecifications required for multiplex analysis: In U.S. Pat. No.4,774,189 Schwartz fluorescent proteins are coupled to particles togenerate standards for flow cytometric analysis using similarfluorescent proteins. It discloses that particles with differentintensities of fluorescence can be generated by covalently attachingfluorescent proteins to particles. However, since these particles weredesigned for standardization purposes, no attempts were made to coupletwo fluorochromes simultaneously to the particles. Furthermore, noattempts were made to attach a capture reagent molecule to the sameparticles. The fluorescent probes used are proteins that denature inharsh conditions such as those used for hybridization of DNA in buffersused for immunoprecipitation that contain denaturing detergents such asSDS. Furthermore, attachment of fluorescent proteins to particles willcompromise the binding of capture reagents such as antibodies to thesame particles. Their method is therefore not applicable for generatingmulticolored particles for multiplex analysis. U.S. Pat. No. 5,194,300Cheung reports small (300 angstrom) fluorescent particle that could beused to enhance signals for detection. The inventors show that it waspossible to generate particles that have a single fluorescence intensityand a capture reagent bound to their surface. It was not reportedwhether binding of the dyes interfere with subsequent binding ofbiomolecules. Moreover, no attempts are made to generate particles withseveral different intensities of fluorescence or to couple two colors tothe same particle.

The second approach to particle dying is represented by U.S. Pat. No.5,073,498 Schwartz and U.S. Pat. No. 4,717,655 Fulwyler. The formerdiscloses two or more fluorescent dyes added during polymerizationprocess and randomly dispersed within the body of the particle. However,when such particles are exposed to a single excitation wavelength onlyone fluorescent signal is observed at a time and thus these particlesare not useful for multiparameter analysis especially in a flowcytometry apparatus with a single excitation light source. U.S. Pat. No.4,717,655 Fulwyler discloses two dyes mixed at five different ratios andcopolymerized into a particle. Although five populations of beads wereclaimed as being obtainable, the fluorescent properties of these beadsare not provided. In conclusion, both U.S. Pat. No. 5,073,498 Schwartzand U.S. Pat. No. 4,717,655 Fulwyler represent complex and costlymethods for producing multicolored particles comprising internalincorporation of dyes.

The principle of the third method, i.e., internally embedding ordiffusing a dye after a particle has been already polymerized wasoriginally described by L. B. Bangs (Uniform Latex J Particles; SeragenDiagnostics Inc. 1984, p. 40) and consists of adding an oil-soluble orhydrophobic dye to stirred microparticles and post-incubation washingoff the dye. The microspheres used in this method are hydrophobic bynature. This allows adopting the phenomenon of swelling of suchparticles in a hydrophobic solvent, which may also contain hydrophobicfluorescent dyes. Once swollen, such particles will absorb dyes presentin the solvent mixture in a manner reminiscent to water absorption by asponge. The level and extent of swelling is controlled by incubationtime, the quantity of cross-linking agent preventing particle fromdisintegration, and the nature and amount of solvent(s). By varyingthese parameters one may diffuse a dye throughout particle or obtainfluorescent dye-containing layers or spherical zones of desired size andshape. Removing the solvent terminates the staining process.Microparticles stained in this manner will not “bleed” the dye inaqueous solutions or in the presence of water-based solvents orsurfactants such as anionic, nonionic, cationic, amphoteric, andzwitterionic surfactants. U.S. Pat. No. 5,723,218 Haugland disclosesdiffusely dyeing microparticles with one or more dipyrrometheneborondifluoride dyes by using a process, which is essentially similar to theBangs method. However, when beads internally stained with two separatedipyrrometheneboron dyes, were excited at 490 nm wavelength, theyexhibited overlapping emission spectra. Hence, the beads weremonochromatic and not multicolored. U.S. Pat. No. 5,326,692 Brinkley etal; U.S. Pat. No. 5,716,855 Lerner et al; and U.S. Pat. No. 5,573,909Singer et al. disclose fluorescent staining of microparticles with twoor more fluorescent dyes. However, dyes used in these processes haveoverlapping excitation and emission spectra allowing energy transferfrom the first excited dye to the next dye and through a series of dyesresulting in emission of light from the last dye in the series. Thisprocess was intended to create an extended Stokes shift, i.e., a largergap between the excitation and emission wavelength, and not the emissionof fluorescence from each dye simultaneously. Thus, due to variousreasons such as dye-dye interaction, overlapping spectra, non-Gaussianemission profiles and energy transfer between neighboring dyes, thedemand for multicolored beads simultaneously emitting fluorescence atdistinct peaks was not satisfied.

U.S. Pat. No. 5,786,219 Zhang devised microspheres with two-colorfluorescent “rings” or microspheres containing a fluorescent spherical“disk” combined with a fluorescent ring. Nevertheless, such beads,designed for calibration purposes, cannot be used in multiparameteranalysis since two dyes were mixed only at one fixed ratio. However, thehighest number of dyes ratios ever attempted with at least two dyesnever exceeded five.

Chandler et al (U.S. Pat. No. 6,599,331) disclose a method that isessentially similar to that disclosed by Bangs and later applied byHaugland, Brinkley and Lerner. The main difference being the choice offluorescent dyes. The inventors were able to find a combination of dyesthat resulted in dual emission from the particles. However, this methodmay be limited to a few selected dyes since previous results by HauglandBrinkley and Lerner showed that energy transfer resulted inmonochromatic emission. In U.S. Pat. No. 6,649,414, Chandler et aldisclose a method where nano-particles are dyed according to the sameprocedure as that disclosed in U.S. Pat. No. 6,599,331. Thesenanoparticles were then attached to the surface of larger polymerparticles to generate a new particle consisting of a core particle and alayer of variable numbers of nano-particles on the surface. Suchparticles will, however, have an irregular surface and therefore highlyvariable light scattering properties and most likely a high tendency foraggregation in solution. In addition, non-specific binding of proteinsfrom e.g. a cell lysate will tend to increase when the surface isirregular.

The following challenging aspects are relevant when developing amulti-colored particle:

1. Since surface labeling occurs via reactive groups on the particle,binding of fluorescent dyes and capture reagents will compete for thereactive groups on the particle. Thus, particles that are first labeledwith different amounts of dyes would not be expected to bind similarlevels of reagent used to bind the analyte. Alternatively, particlesthat have first bound the reagent used to bind the analyte would beexpected to have few groups available for the reactive groups of thefluorescent dyes.

2. Surface labeling with multiple fluorescent compounds might beexpected to lead to a large degree of fluorescence energy transferbetween the dyes. This would greatly limit the number of codes that canbe generated. Color-coding based on two or more fluorescent probesimplies that the emission and, or absorption spectra of the probes aresufficiently different to allow simultaneous independent detection ofthe two probes. When two probes are in close proximity fluorescenceenergy transfer may occur. This implies that the light emitted by one ofthe probes is absorbed by the second and thus quenched. This phenomenonis well known and may occur even between probes that have largedifferences in emission and absorption spectra. An example isPhycoerythrin and Cy.5, where the emission spectrum of Phycoerythrin andthe absorption spectrum of Cy5 is separated by >100 nm. In this case thefluorescence of Phycoerythrin is completely quenched by Cy5. When dyesare incorporated into the polymer, they are distributed throughout thevolume of the particles. The surface area of the particle is a muchsmaller distribution area for the probes. Therefore one might expectthat the probes would be in close proximity. This could limit the numberof measurable color codes to the extent that true multiplex color codingwould be impossible.

3. It is expected that fluorescent dyes bound to the surface ofparticles may interfere with fluorescent signals from the analyte due tofluorescence energy transfer. Thus, if the fluorescent probe used todetect the analyte can transfer energy to the dye used for color-codingor vice versa, one would expect that the analyte signal would bedifferent on particles with different color codes.

4. Furthermore, one would expect that surface labeling is notsufficiently stable to allow discrimination of small differences influorescence when particles are subjected to storage or reactions thatrequire harsh conditions such as high temperatures.

5. Lastly, fluorescent dyes may undergo changes in spectralcharacteristics upon binding to monodisperse latex spheres.

In our opinion, no reliable microsphere populations or subsets emitting,upon exposure to a single excitation wavelength, multiple fluorescentsignals of variable intensity and at spaced, optically distantwavelengths from surface-bound dyes or a combination of internal andsurface-bound dyes have so far still been disclosed. In particular,there is a great need for particles with said characteristics whichfurther permit use of a wide range of commercially available reactiveforms of fluorescent dyes, which are produced by a simple andcost-effective method and which can be dyed after labeling with uniformlevels of a capture reagent.

SUMMARY

The instant invention describes a novel set of polymer particles stainedwith at least two fluorescent dyes, wherein at least 16 subsets ofparticles can be resolved on the basis of variable emission from the atleast two fluorescent dyes wherein emission from at least one dyederives from a fluorescent dye covalently attached to the particlesurface, and wherein all particles in said set of polymer particles canbind a uniform amount of a capture reagent. Surprisingly, the presentinvention shows that it is possible to generate at least 100 differentcolor-codes on particles first and then obtaining highly uniform levelsof a capture reagent. Moreover, the disclosed data show that 100different color codes (subsets of particles) can be generated usingparticles that have first been reacted with a single concentration of acapture reagent. The present invention demonstrates that (1) even thoughfluorescence energy transfer does occur, it is still possible togenerate 100 different color codes (subsets of particles) via surfacelabeling; (2) that fluorescence energy transfer between the probe usedfor detection and that used for color-coding does not occur. Thusparticles that were color-coded by surface labeling with differentamounts of the fluorescent dyes Alexa 647 and Alexa 488 had the sameintensity of phycoerythrin fluorescence when incubated with the sameamount of a phycoerythrin-labeled analyte. This is an unexpected findingsince Alexa 647 is known as a very effective acceptor for phycoerythrin.(3) Present data show that fluorescent properties of the color-codedparticles is unchanged by boiling of the particles; (4) that dyes, likeAlexa 647, undergoes drastic time-dependent changes in their spectralproperties upon binding to monodisperse latex spheres. Quiteunexpectedly, this far-red dye has considerable green fluorescencefollowing storage of particles at 4 C. (5) Finally, the presentinvention demonstrates that changes in spectral characteristics can beavoided by the use of maleimide derivatives of dyes and by storing dyedparticles at 20 C in a cryopreservation medium consisting of PBS with50% trehalose.

Hence, the inventors believe the current invention accommodate theabove-mentioned needs by providing a surface-multicolored set ofparticles, as well as methods and uses thereof, allowing a plurality ofdefined subsets of stained colored microparticles distinguishable by asubtle variation in fluorescence signal resulting from the combinationof various dyes of distinct color and having variable intensity of coloremission. The present surface labeling permits the use of a wide rangeof reactive forms of fluorescent dyes that are commercially available.The labeling process is greatly simplified compared to incorporation ofdyes into the polymer. In addition, it is possible to dye particles thatare already labeled with uniform levels of a capture reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Attachment of crosslinker to beads, priming antibodies, andattaching the antibody to the particle via a crosslinker.

FIG. 2 Particles

FIG. 3 Particles

FIG. 4 Method of producing particles of the invention by employingbiotin instead of a crosslinker

FIG. 5 Multiplex analysis by flow cytometry

FIG. 6 Particles stained with Alexa 647 maleimide were mixed asdescribed, split in 16 equal aliquots and labeled with Alexa 488maleimide.

FIG. 7 A mixture of particles dyed with Alexa 488 and Alexa 647 weresplit in two, one was incubated with SMCC modified Goat anti-mouse IgGFc, the other not. The two were then mixed again, blocked with PBScontaining 10% FCS and 1% Tween 20 and then incubated with a PE-labeledmouse IgG antibody. The results show uniform staining of particles withantibody regardless of their fluorescence properties.

FIG. 8 Mixed particles were boiled for 5 min with 10% SDS to teststability of the fluorescent signals.

FIG. 9 Amino-terminated microspheres were incubated with SPDP, reducedwith TCEP and then incubated with two-fold dilutions of Alexa 647maleimide. Particles were then washed five times. Fluorescence intensityof particles was measured with a FACSCalibur flow cytometer.

FIG. 10 Quantitation of protein levels.

FIG. 11 Analysis of molecular complexes and fusion proteins.

FIG. 12 Analysis of protein phosphorylation.

FIG. 13 Verification that signals to particles represent the intendedtargets of the antibodies on the molecules.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The current invention comprises a set of polymer particles stained withat least two fluorescent dyes, wherein at least 16 subsets of particlescan be resolved on the basis of variable emission from the at least twofluorescent dyes wherein emission from at least one dye derives from afluorescent dye covalently attached to the particle surface, and whereinall particles in said set of polymer particles can bind a uniform amountof a capture reagent.

Said subset of polymer particles is herein defined as a group of polymerparticles having a unique color code that can be resolved on the basisof their variable emission from the at least two fluorescent dyes.Variable emission is herein defined to also include no emission.

Said at least 16 subsets of particles preferably being at least 25 or 36subsets of particles, and even more preferably being at least 48 subsetsof particles.

The number of subsets of particles that may be resolved on the basis ofvariable emission from said at least two fluorescent dyes can bedirectly and positively verified without undue experimentation by e.g.using the method described in example 2.

Uniform amount and/or uniformly labeled is herein defined as an amountor a labeling that does not significantly vary between particles withinthe set, but is even and regular throughout.

The present invention also relates to a said set of polymer particles,wherein said dyes are attached in defined concentrations. Definedconcentrations is herein defined as the amount of each dye that isattached to the particles in a specific subset of particles. The amountof each dye is herein defined to also include no dye.

The present invention also relates to said set of polymer particles,wherein said at least one fluorescent dye that is covalently attached tothe particle surface is covalently attached via a bifunctionalcrosslinker, biotin-streptavidin chemistry or directly to each particle.

The present invention also relates to said set of polymer particles,wherein all particles in said set of polymer particles containsufficient binding sites for uniform covalent attachment of capturereagent. Said binding sites preferably being free SH- or amino-groups.

The present invention also relates to said set of polymer particles,wherein all particles in said set of polymer particles may be uniformlylabeled with a capture reagent, wherein the capture reagent optionallyhas attached fluorescent dyes. Said capture reagent may benon-covalently attached to the particles. Preferably, said capturereagent may be covalently attached via a bifunctional crosslinker,biotin-streptavidin chemistry or directly to each particle. Said capturereagent may be any molecule capable of interacting with a molecule ofinterest, e.g. a protein in a biological assay. Said capture reagent maybe a protein, e.g. antibody-based molecule or a nucleic acid e.g. DNA orRNA.

The bifunctional crosslinker may be any suitable crosslinker, preferablythe crosslinkers according to Table 1, more preferably, a crosslinkerbased on maleimide-thiol chemistry. The crosslinker may be homo- orheterobifunctional, however, heterobifunctional crosslinkers arepreferred. In an embodiment of the current invention the crosslinker isSPDP.

Suitable fluorescent dyes or fluorochromes, hereafter named dyes, areknown within the art and examples are listed in Table 2. In a preferredembodiment of the current invention the dyes may be UV/violet excitable,488 nm excitable, 532 nm (YAG) excitable, 595 nm (Krypton) excitable,633 nm excitable, Infrared excitable, 488 nm excitable, 633 nm excitableTandem conjugates of PE and APC, Tandem conjugates of reactive dyes(e.g. Alexa dyes) 488 nm excitable and/or quantum dots. Even morepreferably the fluorescent dyes may be hydrophilic forms of cyanine dyessuch as reactive forms of Alexa 488 and 647.

Polymer particles suitable as a starting material for the currentinvention is known in the art and can be obtained from commercialmanufacturers. The initial particles may be formed of e.g. polyvinylchloride, polyvinyl toluene, styrene, or methymethacrylate withpolyvinyl toluene and the particles are preferably less than 100 μm indiameter.

Other examples of microspheres are brominated polystyrene, polyacrylicacid, polyacrylonitrile, polyacrylamide, polyacrolein, polybutadiene,polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate,polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride,polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene,polymethylmethacrylate, or combinations thereof.

The present invention also relates to said set of polymer particles,wherein the particles may be monodispersed particles. Monodisperseparticle is herein defined as a particle with only one molecular mass.

The set of particles of the invention further comprise severalfluorescent dyes or fluorochromes, preferably at least two fluorescentdyes or fluorochromes, and most preferred two fluorescent dyes orfluorochromes.

Dyed nanoparticles are not regarded as fluorochromes in the presentdisclosure.

The surface of the particles, not the core, should be dyed in definedconcentrations.

A certain absorption of dye radially within the particle's surface mayoccur. The above particles render possible heterogenous set of polymerparticles with particle subsets emitting unique combination offluorescent light and carrying a particular capture reagent, e.g. anantibody.

Another aspect of the current invention is a method for the preparationof the set of polymer particles according to the invention comprising,in either sequence:

-   -   Attaching at least two fluorescent dyes in defined        concentrations to the set of polymer particles according to the        invention, wherein at least one dye is covalently attached to        the particle surface, and    -   optionally attaching different capture reagents of interest to        different subsets of polymer particles, wherein said capture        reagent may be covalently attached directly to each particle or        via a bifunctional crosslinker or biotin-streptavidin chemistry        and optionally has attached fluorescent dyes.

Preferably, the method is executed as described in the disclosedexamples of this application.

Another aspect of the current invention is a set of polymer particlesprepared according to the above method.

The set of polymer particles of the invention may be used for specificbinding of biomolecules, including native and modified forms ofpolypeptides and polynucleotides, and allows parallel analysis of amultitude of analytes. For example, the phosphorylation status of amultitude of proteins can be assessed in parallel by means of theparticles of the invention and a flow cytometer. Accordingly, a furtheraspect of the current invention is the use of the set of particlesaccording to the invention in multiplex analysis and/or in the field ofdiagnostics.

A further aspect of the present invention is the set of polymerparticles according to the invention for use in multiplex analysisand/or for diagnostic use.

A further aspect of the present invention relates to a diagnostic methodor a multiplex analysis method comprising the following steps:

(a) attaching different capture reagents of interest to differentsubsets of polymer particles, wherein each subset of polymer particlesthen having a specific capture reagent attached.

(b) mixing said set of polymer particles with a sample of interest

(c) analyzing said set of particles by flow cytometry.

A further aspect of the invention is a kit comprising the set ofparticles according to the invention.

EXAMPLES

1. General Considerations

1.1 Attachment of a Capture Reagent to the Particles

This is performed by incubation of particles with the same concentrationof a capture reagent. Covalent binding is best achieved by firstincubating particles with a crosslinker (Table I). The preferred methodis to use maleimide-thiol chemistry. The heterobifunctional crosslinkerSPDP is attached to amino-derivatized particles and the pyridyldisulfidegroup is reduced with TCEP or DTT. A maleimide group is attached to thecapture reagent (e.g. an antibody or protein A or protein G) byincubation with another heterobifunctional crosslinker such as SMCC(Table I). The modified reagent is then incubated with the particles atpH5.

Alternatively, maleimide groups can be added to the particles with thecrosslinker SMCC. In this case the protein (modified with SPDP or not)is reduced with TCEP at pH5.0 to generate free —SH groups. The resultsare similar. Other types of homo- and hetero-bifunctional crosslinkingagents have been described and would be expected to provide similarresults. (Table I)

Finally, proteins such as antibodies may be bound to particles byphysical adsorption. This does not provide covalent coupling. However,for several applications, the binding obtained by physical adsorption issufficiently stable. This is a routinely used way to couple proteins tolatex in assays such as ELISA.

1.2. Color-Coding

Color coding is performed by incubation of particles with definedconcentrations of reactive forms of two or more fluorescent dyes. Bounddye is separated from unbound dye by centrifugation of particles,removal of supernatant and the particles are suspended in a new buffer.Color coding can be performed after binding of a general capture reagentsuch as anti-mouse IgG. Alternatively, particles can be dyed first andthen coupled to a capture reagent at a later time point. In most caseshigher concentrations of dyes are necessary when color coding isperformed after the binding of a capture reagent.

Example 2

The preferred method is the use of maleimide-derivatives of dyes. Thesedyes bind to free —SH groups that remain following attachment of thecapture reagent (FIG. 2). Preferred combinations are dyes with largedifferences in emission spectra such as Alexa 647 and Alexa 488. Dyesare dissolved at 1:1,75 fold dilutions in MES buffer pH6.0 usuallystarting at 100-1000 ng/ml of dye. Particles with free —SH groups (seeprocedure for attaching capture reagents) are resuspended in MES bufferpH6. This procedure was followed and the batch was first split in 12equal aliquots. Each was incubated with a different concentration of onemaleimide dye at 37° C. with frequent mixing for 15 min and then, cooledto 4° C. and washed. Each aliquot was then split in 8 new aliquots. Eachof the new aliquots was incubated with different concentrations of thesecond fluorescent dye. FIG. 6 shows that 96 different color codes weremade by this procedure. FIG. 7 shows that all particles bind similaramounts of a capture reagent.

Example 3

The method can be extended to other reactive forms of fluorescent dyes.Amine-reactive forms such as N-hydroxy-succimidyl esters (NHS esters)bind to free amines on the capture reagent or amines that were not usedfor attachment of the crosslinker (FIG. 3). The reaction is performed atpH7.4 or higher.

Example 4

The method can be extended to using biotin instead of a crosslinker onthe particle (FIG. 4). The particles are first incubated with reactiveforms of biotin, then with variable concentrations of astreptavidin-fluorophore-conjugate such as streptavidin-Phycoerythrin,then with variable concentrations of a secondstreptavidin-fluorophore-conjugate such as Streptavidin PerCP. Finally,the particles are reacted with saturating concentrations of unlabeledstreptavidin. The particles are finally reacted with a biotinylatedcapture reagent.

Example 5

Detailed Description of the Method to Generate a Multiplex withAnti-Mouse Igg as a General Capture Reagent

a. Method for Coupling Antibodies and Dyes to Aminobeads.

Protein Coupling:

Materials:

Aminobeads, 7.74 um or 3.69 um from Bangs Laboratories. Stock: 10%solids stored at 4 C. Beads have free aminogroups on surface.

Crosslinkers: SPDP: one NHS arm that reacts with aminobeads, onedisulphide arm that can be reduced and thus provides free —SH groups onbeads.

Sulfo-SMCC: one NHS arm that binds to aminogroups on antibodies, onemaleimide arm that binds to thiols on beads. Both crosslinkers arestored at −70 in DMSO the concentration of the stock is 10 mg/ml.

TCEP: a strong reducing agent, prepare a 100 mM solution in water beforeeach experiment. (powder stored at room temp together with dry chemicalsin our lab). Typically weigh out 3 mg in an eppendorf tube and add 100ul water, mix well.

Buffers: PBS with 1% tween and 5 mM EDTA., MES 100 mM pH5.2, 100 mM MESpH6 with 1% tween and 5 mM EDTA.

Tubes: 15 ml polypropylene tubes, 1.5 ml Eppendorf tubes.

Gels for buffer exchange of proteins: Sepharose G50 fine, add 3.5 gpowder to a 50 ml tube, fill with each buffer. Mix well and let itsettle before use to avoid bubbles.

Columns for Buffer Exchange of Proteins:

Microspin columns: can take 1 ml of gel, use for samples less than 100ul Biospin columns: can take 2 ml of gel, use for samples up to 200 ulPD-10 columns: can take 10 ml of gel and are used up to 1.5 ml sample.

Use of Columns:

Microspin: add gel, place column on an eppendorf tube, cut off lid, spinfirst for 10 sec in the microfuge, discard fluid from Eppendorf tube.Place column on the eppendorf tube again and centrifuge for 30 sec. Thecolumn is now ready for use. Biospin: add gel, place column on a flowcytometry tube, centrifuge for 5 min at 1600 rpm. Vacuum away fluid fromtube. The column is now ready for use.

PD-10: Add gel, place column on an eppendorf tube inside a 50 ml tube,centrifuge for 5 min at 1600 rpm. Discard the tubes, and place thecolumn in new Eppendorf/50 ml tube. It is now ready for use.

Add sample to the center of the gel. Add 10% extra volume on top withwanted buffer. Spin 5 min 1600 rpm for Biospin and PD10, 30 sec inmicrofuge for microspin. The protein is in the tube at the bottom in thenew buffer.

b. Procedure for Making an Array from 1 ml of Aminobeads.

Part 1. Coupling of Proteins to Beads.

1. Take out 0.5 mg protein, if it is in PBS, use as is, if not: bufferexchange on G-50 PBS, add 10 ul of SMCC stock per 1 ml of proteinsolution. Mix well and leave on the bench for 30 min-1 h (basically toall other steps of part 1 are done)

2. Add 1.5 ml Aminobeads to a 15 ml tube filled with 13 ml withPBS-Tween EDTA, centrifuge for 3 min at 1200 rpm.

2. Discard supernatant, whirlmix the pellet well and add 0.5 ml of PBStween. Resuspend with a 1 ml pipette. Adjust volume to 10 ml and add 100ul SPDP stock, mix well and rotate for 30 min at room temp.

3. Wash twice with PBS tween, adjust volume to 2 ml

4. Add 20 ul 100 mM TCEP, mix well and incubate 15 min at 37 C waterbath, whirlmix every 5 min

Wash with MES pH 5.2

Resuspend beads by adding 0.5 ml MES pH5.2 and whirlmix, adjust volumeto 1 ml, sonicate, one shot only.

5. Buffer exchange protein into MES pH5.2.

6. Dissolve the protein in 5 ml MES pH5.2 to obtain a concentration of100 ug/ml

Add beads to antibody solution and rotate for 24 h at room temp.

After 24 h of coupling:

Wash beads three times in MES ph5.2

Resuspend in 1 ml MES pH6

Proceed to color-coding protocol.

c. Protocol for Color Coding.

Materials:

Alexa 488 maleimide, stock 10 mg/ml in DMSO stored at −70 C

Alexa 647 maleimide stock 10 mg/ml in DMSO stored at −70 C

Buffers: MES pH6.0 with 5 mM EDTA and 1% Tween 20. PBS with 5 mM EDTAand 1% Tween 20.

Tubes: 15 ml Polypropylene tubes, 1.5 ml eppendorf tubes, 1 ml Bioradtiter tubes in 96 well rack format.

Procedure:

Beads with thiol groups on (see part 1 of protocol) are suspended in1-1.5 ml at 10% solids in MES pH6 with EDTA and tween before start.

First part: find the starting concentration for Alexa 647 (variableamounts of —SH groups are consumed when coupling proteins, therefore, wefirst need to determine how much maleimide dyes we need to add to get alinear decrease in fluorescence with serial dilutions of the dye.

1. Prepare solution of Alexa 647 10 ug/ml by adding 1 ul stock to 1 mlMES pH6.

2. Prepare 10 fold dilutions, i.e. 1 ug, 100 ng, 10 ng and 1 ng, ineppendorf tubes.

3. Add 1 ul of the bead suspension to each tube, incubate 10 min inwater bath, mix at least twice during the incubation period.

4. Spin tubes, add MES and run flow cytometry.

5. Choose starting concentration for Alexa 647 that is to be used onparticles. The starting concentration should be the highest, where alinear signal can be expected.

Thus, if there is a 10 fold difference in fluorescence of 0.1 and 1 ug,and only a 2 fold between 1 and 10 ug, the linear signal starts at 1 ug.Usually, go a little lower for example 200 ng.

Second part: Determine exactly which concentrations are useful for colorcoding.

6. Set up 16 15 ml tubes. Label from 1-20. Add 14 ml MES pH6 to thefirst with a 50 ml pipette and 6 ml to the rest.

7. Adjust the Alexa 647 concentration of tube 1 to the chosen startingconcentration.

8. Add 8 ml from tube 1 to tube 2, put on lid and mix well.

9. Repeat this to tube 15, leave tube 16 without any dye. Leave thetubes in wet ice.

10. Add 1 ul bead suspension to each of 16 1.5 ml eppendorf tubes.

11. Add 100 ul of each dye dilution to each tube. Mix well and incubate15 min at 37 C, mix every 5 min.

12. run Flow cytometry.

13. Choose 11 different concentrations that provide fluorescence signalsthat can be resolved and one blank.

14. Make sure beads are suspended in at least 1250 ul. Add 100 ul toeach of the 12 chosen 15 ml tubes. Mix the tube immediately after addingthe beads.

15. Place tubes in 37 C water bath. Mix every 5 min, incubate for 20min.

16. Place tubes in ice-water, leave for 5 min to cool down. Spin down inrefrigerated centrifuge at 4 C.

17. Wash twice in MES pH6. resuspend in MES pH6. 900 ul.

The beads can now be left in the fridge overnight if necessary.

Second dye: Usually Alexa 488 maleimide.

1. Prepare a 10 ug/ml Alexa 488 maleimide solution in MES pH6 by adding1 ul stock to 1 ml buffer in an eppendorf tube. Make four 10 folddilutions, i.e. 1 ug, 100 ng, 10 ng and 1 ng.

2. Prepare a mix of the 12 beads that were labeled with Alexa 647 byadding 10 ul of each to an eppendorf tube.

3. Adjust volume to 200 ul.

4. Add 10 ul to each of the 4 dilutions of Ax 488 in eppendorf tubes,mix and place in water bath for 15 min with shaking every 5 min.

5. spin, resuspend in MES pH6 and run FACS., find starting dilution forAx 488. (see previous section for Ax 647).

6. Set up 15 15 ml tubes. Add 15 ml MES to the first and 5 ml to therest.

7. Adjust Ax488 to the starting conc. In the first tube.

8. Take out 10 ml and add to tube #2. and continue to tube 15.

9. Leave tubes in ice.

10. Add each of the 12 Ax 647 bead aliquots to titer tubes, place1,3,5,7,9,11 in the first rack and 2,4,6,8,10,12 in the second. Theseare referred to as rows.(i.e. all tubes in each row have the sameconcentration of Ax 647)

11. Fill up the rows to total 8 columns in each rack. 11. Using an 8channel pipette, take out 50 ul and add to each of 8 titer tubes in therack.

12. Leave racks on ice.

13. Using an Eppendorf stepper, add 500 ul of ice cold Ax 488 maleimidedilution to each. Dilution 1,3,5,7,9,11,13,15 to the first rack,dilutions 2,4,6,8,10,12,14 and blank to the second.

14. Shake tubes individually, put back on ice after shaking.

15. Place racks in a Styrofoam box with 37 C water in the bottom so thatthe interior of the rack is filled with water, (be careful!)

16. Mix tubes almost continuously for 15 min. Be careful not to spill.

17. after 15 min, centrifuge the racks and wash four times with coldMES. Finally resuspend in PBS tween. (we will determine whether it isuseful to add NEM 1 mM during the last washing step with MES to quenchfree sulfhydryls.)

Example 6

Antibody-coupled particles can be used to detect differences in proteinexpression between cancer cells and normal cells. This application couldhave diagnostic utility for example in detecting upregulated oncogenicproteins in cancer cells.

Quantitation of Protein Levels.

Cell lysates from an erythroleukemia cell line (K562) and normalleukocytes were labeled with amino-reactive forms of biotin anddigoxigenin, respectively. The lysates were mixed and incubated with amixture of color-coded particles that were coupled with antibodies tocellular proteins. The cartoon to the left in FIG. 10 illustratesbinding of labeled molecules from the two cell types to a particle. Inthis case leukocytes (grey) express two-fold more of the protein thanthe leukemic cells (dark grey). The flow cytometry plot next to thecartoons data in FIG. 10 obtained with antibodies to leukocyte surfacemarkers (two left diagrams). Particles that mainly capture proteins fromleukemia cells are represented as dark grey dots, whereas those thatcapture from leukocytes are grey. The positions in the array are shownin to the right in FIG. 10. The two diagrams to the right in FIG. 10show similar analysis for antibodies to signaling proteins. Light greydots represent particles that bind proteins from both cell types.

Analysis of Molecular Complexes and Fusion Proteins.

Detection of molecular complexes may have diagnostic utility for exampleby revealing dysregulated cell signaling pathways.

A mixture of particles with antibodies to signaling proteins was firstincubated with unlabeled cell proteins and then labeled with antibodiesto the adaptor protein grb-2 (cartoon in FIG. 11). Anti-grb2 binds toparticles that have captured grb2 either directly with a specificantibody or indirectly via molecules that associate with grb2. Flowcytometric measurement showed that grb-2 was associated with 6 of 96proteins (populations on the right of the plot in FIG. 11), among thesewere SHC, PI3 kinase and paxillin, all known to be associated with grb2.The same principle can be used to detect of fusion proteins such as thebcr-abl fusion protein in chronic myeloid leukemia. This shows thatprotein associations can be analyzed by use of color-coded particles.

Analysis of Protein Phosphorylation.

Protein phosphorylation plays a key role in cell biology and isfundamental for the growth of cancer cells. FIG. 12 shows binding of ananti-phosphotyrosine antibody (x axis) to color-coded particles thatwere first incubated with a lysate of stimulated K562 erythroleukemiacells. The particle populations that are shown to the right of thevertical line capture proteins that were phosphorylated on tyrosine. Themolecules include several known tyrosine kinases (c-abl, lck, ntk, yes,fak).

Verification that Signals to Particles Represent the Intended Targets ofthe Antibodies on the Molecules

Color-coded particles with antibodies to leukocyte surface proteins wereincubated with fluorescently labeled leukocyte lysates. The particleswere boiled and the captured proteins resolved by SDS PAGE. The gel wasscanned by a fluorescence scanner. The results show single bands withmolecular weight corresponding to the intended targets of the antibodies(FIG. 13). These results show that the technology correctly measures theintended targets of the antibodies.

TABLE 1 Spacer Arm Water Membrane Acronym PrdNum Length Links CleavableBy Soluble Permeable Homobifunctional crosslinker chemistry EGS 2156516.1 Å Amines To Hydroxylamine No Yes Amines Sulfo-EGS 21566 16.1 ÅAmines To Hydroxylamine Yes No Amines BSOCOES 21600 13 Å Amines To BaseNo Yes Amines DSP 22585 12 Å Amines To Thiols No Yes Amines DTSSP 2157812 Å Amines To Thiols Yes No Amines DTBP 20665 11.9 Å Amines To ThiolsYes Yes Amines DSS 21555 11.4 Å Amines To non No Yes Amines BS³ 2158011.4 Å Amines To non Yes No Amines DMS 20700 11 Å Amines To non Yes YesAmines DMP 21666 9.1999998 Å Amines To non Yes Yes Amines DMA 206638.6000004 Å Amines To non Yes Yes Amines DSG 20593 7.6999998 Å Amines Tonon No Yes Amines MSA 22605 7.1999998 Å Amines To non No nd AminesSulfo-DST 20591 6.4000001 Å Amines To Periodate Yes No Amines DST 205896.4000001 Å Amines To Periodate No Yes Amines DFDNB 21525 3 Å Amines Tonon No Yes Amines Thiol-maleimide chemistry SMPT 21558 20 Å Amines ToThiols No Yes Sulfhydryls Sulfo-LC-SMPT 21568 20 Å Amines To Thiols YesNo Sulfhydryls LC-SMCC 22362 16.1 Å Amines To non No Yes SulfhydrylsKMUA 22211 15.7 Å Amines To non No nd Sulfhydryls Sulfo-KMUS 21111 15.7Å Amines To non Yes No Sulfhydryls Sulfo-LC-SPDP 21650 15.6 Å Amines ToThiols Yes No Sulfhydryls LC-SPDP 21651 15.6 Å Amines To Thiols No YesSulfhydryls SMPB 22416 14.5 Å Amines To non No Yes SulfhydrylsSulfo-SMPB 22317 14.5 Å Amines To non Yes No Sulfhydryls SMPH 22363 14.3Å Amines To non No nd Sulfhydryls Sulfo-SMCC 22322 11.6 Å Amines To nonYes No Sulfhydryls SMCC 22360 11.6 Å Amines To non No Yes SulfhydrylsSIAB 22329 10.6 Å Amines To non No Yes Sulfhydryls Sulfo-SIAB 22327 10.6Å Amines To non Yes No Sulfhydryls Sulfo-GMBS 22324 10.2 Å Amines To nonYes No Sulfhydryls GMBS 22309 10.2 Å Amines To non No Yes SulfhydrylsMBS 22311 9.8999996 Å Amines To non No Yes Sulfhydryls Sulfo-MBS 223129.8999996 Å Amines To non Yes No Sulfhydryls Sulfo-EMCS 22307 9.3999996Å Amines To non Yes No Sulfhydryls EMCA 22306 9.3999996 Å Amines To nonYes No Sulfhydryls EMCS 22308 9.3999996 Å Amines To non No YesSulfhydryls BMPS 22298 6.9000001 Å Amines To non No Nd Sulfhydryls SPDP21857 6.8000002 Å Amines To Thiols No Yes Sulfhydryls SBAP 223396.1999998 Å Amines To non No Yes Sulfhydryls BMPA 22296 5.9000001 ÅAmines To non Yes No Sulfhydryls AMAS 22295 4.4000001 Å Amines To non Nond Sulfhydryls SATP 26100 4.0999999 Å Amines To non No Yes SulfhydrylsSIA 22349 1.5 Å Amines To non No nd Sulfhydryls Carbonyldiimidechemistry AEDP 22101 9.5 Å Amines To Thiols Yes No Carboxyls EDC 22980 0Å Amines To non Yes No Carboxyls Photo-affinity chemistry SASD 2771618.9 Å Amines To Thiols Yes No Nonselective SAND 21549 18.5 Å Amines ToThiols Yes No Nonselective SANPAH 22600 18.200001 Å Amines To non No YesNonselective Sulfo-SANPAH 22589 18.200001 Å Amines To non Yes NoNonselective Sulfo-NHS-LC- 27735 18 Å Amines To non Yes No ASANonselective SADP 21533 13.9 Å Amines To Thiols No Yes NonselectiveSulfo-SADP 21553 13.9 Å Amines To Thiols Yes No Nonselective Sulfo-HSAB21563 9 Å Amines To non Yes No Nonselective NHS-ASA 27714 8 Å Amines Tonon No Yes Nonselective ANB-NOS 21451 7.6999998 Å Amines To non No NoNonselective TFCS 22299 7.6999998 Å Amines To non Yes nd NonselectiveSulfo-SBED 33033 Å Amines To Thiols Yes No Nonselective SPB (NHS- 23013Å Amines To non No Yes Psoralen) Nonselective Hydrazine-benzaldehydechemistry SANH convertion of amines to aldehydes SHTH convertion ofamines to aldehydes SFB convertion of amines to benzaldehydes Biotinchemistry Biotin-derivatives Biotin NHS Biotin maleimide Biotin TFPester Biotin-BMCC Sulfhydryl 32.599998 Å No No Yes Biotin-HPDPSulfhydryl 29.200001 Å Yes No Yes PEO-Maleimide Sulfhydryl 29.1 Å No YesNo Activated Biotin Iodoacetyl-LC- Sulfhydryl 27.1 Å No No Yes BiotinPEO-Iodacetyl- Sulfhydryl 24.700001 Å No Yes No Biotin Spacer ChemicalArm Water Membrane Product Name Reactivity Length Cleavable SolublePermeable Biotin-PEO-LC-Amine Carboxyl 22.9 Å No Yes No Biotin-PEO-AmineCarboxyl 20.4 Å No Yes No 5-(Biotinamido)-pentylamine Carboxyl    Å NoYes No

TABLE 2 Commonly used fluorochromes UV/violet excitable Alexa 350, Alexa405, Alexa 430, cascade blue, cascade yellow, 488 excitable fluorescein,alexa 488, bodipy, R-Phycoerythrin, PerCP, 532 (YAG) excitable Cy3,Alexa 547, dylight 547, R-phycoerythrin, B-phycoerythrin, Oyster 550,Oyster 556, Atto 520, Atto 532, atto 595 (Krypton) excitable Texas red,Alexa 610, 633 excitable Cy5, Cy5.5, Alexa 610, Alexa 633, Alexa 647,Alexa 680, Allophycocyanin, Oyster 645, Oyster 650, Oyster 656 Infraredexcitable Alexa 700, Alexa 750, atto 680, Tandem conjugates of PE andAPC (PE = phycoerythrin, APC = allophycocyanine) 488 excitable PE-Cy5,PE, Cy5.5, PE Alexa 610, PE Texas red, PE-Alexa 680, PE Alexa 633, PerCPCy5-5 633 excitable APC, APC-Cy7, Tandem conjugates of Alexa dyes 488excitable DyeMer 488-605, DyeMer 488-615, DyeMer 488-630 quantum dotsLake placid Blue, Adirondack green, Catskill green, Hops Yellow, BirchYellow, Fort Orange, Adams Apple red,

1. A set of polymer particles stained with at least two fluorescentdyes, wherein at least 16 subsets of particles can be resolved on thebasis of variable emission from the at least two fluorescent dyes,wherein emission from at least one dye of the at least two fluorescentdyes derives from a fluorescent dye covalently attached to a particlesurface of at least one particle in the set of polymer particles,wherein all particles in said set of polymer particles can bind auniform amount of a capture reagent, and wherein the at least twofluorescent dyes are not dyed nanoparticles.
 2. The set of polymerparticles of claim 1, wherein said dyes are attached in definedconcentrations.
 3. The set of polymer particles according to claim 1,wherein said at least one fluorescent dye that is covalently attached tothe particle surface is covalently attached via a bifunctionalcrosslinker or directly to each particle.
 4. The set of polymerparticles according to claim 1, wherein all particles in said set ofpolymer particles contain sufficient binding sites for uniform covalentattachment of capture reagent.
 5. The set of polymer particles of claim4, wherein said binding sites are free SH- or amino-groups.
 6. The setof polymer particles according to claim 1, wherein all particles in saidset of polymer particles are uniformly labeled with said capturereagent.
 7. The set of polymer particles of claim 6, wherein saidcapture reagent is covalently attached via a bifunctional crosslinker ordirectly to each particle.
 8. The set of polymer particles according toclaim 1, wherein the capture reagent optionally has attached fluorescentdyes.
 9. The set of polymer particles according to claim 1, wherein saidcapture reagent is a protein or a nucleic acid.
 10. The set of polymerparticles according to claim 9, wherein said protein is anantibody-based molecule.
 11. The set of polymer particles according toclaim 3, wherein the bifunctional crosslinker is based onmaleimide-thiol chemistry.
 12. The set of polymer particles according toclaim 11, wherein the bifunctional crosslinker is SPDP.
 13. The set ofpolymer particles according to claim 1, wherein the fluorescent dyes areUV/violet excitable, 488 nm excitable, 532 nm (YAG) excitable, 595 nm(Krypton) excitable, 633 nm excitable, Infrared excitable, 488 nmexcitable, 633 nm excitable Tandem conjugates of PE and APC, Tandemconjugates of reactive dyes, 488 nm excitable and/or quantum dots. 14.The set of polymer particles according to claim 1, wherein thefluorescent dyes are hydrophilic forms of cyanine dyes such as reactiveforms of Alexa 488 and
 647. 15. The set of polymer particles accordingto claim 1, wherein the size of each particle is less than 100 μm indiameter.
 16. The set of polymer particles according to claim 1, whereinthe particles are monodispersed particles.
 17. A method for thepreparation of the set of polymer particles according to claim 1comprising, in either sequence: a. attaching the at least twofluorescent dyes in defined concentrations to the set of polymerparticles, wherein at least one dye is covalently attached to theparticle surface; and b. optionally attaching different capture reagentsof interest to different subsets of polymer particles, wherein saidcapture reagent may be covalently attached directly to each particle orvia a bifunctional crosslinker or biotin-streptavidin and optionally hasattached fluorescent dyes.
 18. A set of polymer particles preparedaccording to the method of claim
 17. 19. A kit comprising a set ofpolymer particles according to claim
 1. 20. The set of polymer particlesaccording to claim 1, wherein said set of polymer particles is stainedwith two fluorescent dyes.